Lohithakshan Anusree, Narayanasamy Raja, Deshmukh Prashant, Usharani Dandamudi, Kumar Ravi
Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute (CFTRI), Mysore, Karnataka 570020, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Department of Food Safety and Analytical Quality Control Laboratory, CSIR-Central Food Technological Research Institute (CFTRI), Mysore, Karnataka 570020, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Biochim Biophys Acta Proteins Proteom. 2022 May 1;1870(5):140781. doi: 10.1016/j.bbapap.2022.140781. Epub 2022 Apr 11.
The bifunctional flavin adenine dinucleotide synthetase (FADS) synthesizes the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) co-factors essential for the function of flavoproteins. The Staphylococcus aureus FADS (SaFADS) produces FMN from riboflavin (RF) by ATP:riboflavin kinase (RFK) activity at its C-terminal domain. The N-terminal domain converts FMN to FAD under a reducing environment by FMN:ATP adenylyltransferase (FMNAT) activity which is reversible (FAD pyrophosphorylase activity). Herein, we investigated the role of F26 residue of the 24-GFFD-28 motif of SaFADS FMNAT domain, mostly conserved in the reducing agent-dependent FADSs. The steady-state kinetics studies showed changes in the K values for mutants, indicating that the F26 residue is crucial for the FMNAT activity. Further, the FMNAT activity of the F26S mutant was observed to be higher than that of the wild-type SaFADS and its other variants at lower reducing agent concentration. In addition, the FADpp activity was inhibited by an excess of FAD substrate, which was more potent in the mutants. The altered orientation of the F26 side-chain observed in the molecular dynamics analysis suggested its plausible involvement in stabilizing FMN and ATP substrates in their respective binding pockets. Also, the SaFADS ternary complex formed with reduced FMN exhibited significant structural changes in the β4n-β5n and L3n regions compared to the oxidised FMN bound and apo forms of SaFADS. Overall, our data suggests the functional role of F26 residue in the FMNAT domain of SaFADS.
双功能黄素腺嘌呤二核苷酸合成酶（FADS）可合成黄素蛋白功能所必需的黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）辅因子。金黄色葡萄球菌FADS（SaFADS）在其C端结构域通过ATP：核黄素激酶（RFK）活性从核黄素（RF）产生FMN。在还原环境下，N端结构域通过FMN：ATP腺苷酸转移酶（FMNAT）活性将FMN转化为FAD，该活性是可逆的（FAD焦磷酸化酶活性）。在此，我们研究了SaFADS的FMNAT结构域中24 - GFFD - 28基序的F26残基的作用，该残基在依赖还原剂的FADS中大多保守。稳态动力学研究表明突变体的K值发生了变化，表明F26残基对FMNAT活性至关重要。此外，在较低还原剂浓度下，观察到F26S突变体的FMNAT活性高于野生型SaFADS及其其他变体。此外，过量的FAD底物会抑制FADpp活性，在突变体中这种抑制作用更强。分子动力学分析中观察到的F26侧链方向改变表明其可能参与稳定FMN和ATP底物在各自结合口袋中的状态。而且，与结合氧化型FMN和apo形式的SaFADS相比，与还原型FMN形成的SaFADS三元复合物在β4n - β5n和L3n区域表现出显著的结构变化。总体而言，我们的数据表明F26残基在SaFADS的FMNAT结构域中具有功能作用。
